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Structure and chromosomal localization of the mouse bombesin receptor subtype 3 gene
Gene 211 (1998) 125–131
Structure and chromosomal localization of the mouse bombesin receptor subtype 3 gene H.C. Weber a,*, L.L. Hampton b, R.T. Jensen c, J.F. Battey b a Section of Gastroenterology, Boston University School of Medicine, 88 East Newton Street, Boston, MA 02118-2393, USA b National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, MD 20850, USA c Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA Received 3 July 1997; received in revised form 30 January 1998; accepted 2 February 1998; Received by A. Dugaiczyk
Abstract Bombesin (BN )-like peptides/neurotransmitters mediate a broad range of physiological funtions in the gastrointestinal tract and the central nervous system through binding to their specific, high-affinity mammalian bombesin receptors. This family of heptahelical, G protein-coupled receptors includes the gastrin-releasing peptide receptor (GRP-R, or bb2), neuromedin B receptor (NMB-R, or bb1), and the bombesin receptor subtype 3 (BRS-3, or bb3). The tissue distribution of BRS-3 is quite dissimilar compared to the other two BN receptors, GRP-R and NMB-R, and a natural ligand for BRS-3 is currently unknown. Nothing is known about mechanisms regulating BRS-3 gene expression and possible association with disease. To gain insight into the underlying structure and chromosomal localization of the BRS-3 genes, bacteriophage P1 genomic clones, harboring the genes for the human and mouse BRS-3, respectively, were isolated and their structure and chromosomal localizations determined. The protein-coding region of both genes is divided into three exons and spans approximately 5 kb. The loci of the BRS-3 genes were mapped to a syntenic region of the human ( Xq25) and mouse ( XA7.1–7.2) X-chromosome, respectively. The structural data of the BRS-3 genes derived from this study will permit future investigations of the mechanisms regulating their expression. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Gene structure; Mouse X-chromosome; Human X-chromosome; Genomic clones; Mammalian; G protein-coupled receptors
1. Introduction The bombesin (BN )-like peptides/neurotransmitters mediate a variety of biological activities in the central nervous system (CNS) and gastrointestinal (GI ) tract, including smooth muscle contraction and secretion of other GI hormones (Lebacq-Verheyden et al., 1990; Severi et al., 1991; Ghatei et al., 1982). They have also been implicated as autocrine growth factors in the * Corresponding author. Tel: +1 617 638 6547; Fax: +1 617 638 7785; e-mail: [email protected] Abbreviations: BN, bombesin; bp, base pair(s); BRS-3, bombesin receptor subtype 3; DAPI, 4∞, 6-diamidino-2-phenylindole; FISH, fluorescence in-situ hybridization; GRP, gastrin-releasing peptide; GRP-R, gastrin-releasing peptide receptor; kb, kilobase pairs; NMB, neuromedin B; NMB-R, neuromedin B receptor; PCR, polymerase chain reaction; PHA, phytohaemagglutinin; SCLC, small cell lung carcinoma. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 05 0 - X
pathogenesis and progression of some human small cell lung carcinomas (Cuttitta et al., 1985) and the stimulation of proliferation of breast, colon, gastric, small cell lung cancer and prostate cancer cells in vitro or in vivo ( Weber et al., 1985; Carney et al., 1987; Alexander et al., 1988; Bologna et al., 1989; Nelson et al., 1991; Narayan et al., 1992; Bold et al., 1994). The amidated tetradecapeptide bombesin (BN ) has been isolated originally from the skin of the European frog Bombina bombina ( Erspamer et al., 1972), and to date, two mammalian BN-like peptides, the gastrin-releasing peptide (GRP) and neuromedin B (NMB), have been isolated and characterized ( Erspamer, 1988). BN-like peptides mediate their wide spectrum of functions by binding to specific, high-affinity cell surface receptors that belong to the superfamily of heptahelical G protein-coupled receptors. The receptors for the mammalian peptides GRP and NMB have been characterized, and their cDNAs have been isolated from human and murine
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tissues (Spindel et al., 1990; Battey et al., 1991; Corjay et al., 1991). Much less is known about the third member of this receptor family, the bombesin receptor subtype-3 (BRS-3) whose natural ligand is unknown. Human BRS-3 cDNA was isolated from a small cell lung carcinoma (SCLC ) cell line, and is expressed in a panel of human lung carcinoma cell lines (Fathi et al., 1993), and in the pregnant human myometrium (Gorbulev et al., 1994). In addition, BRS-3 mRNA was also expressed at a very low abundance in the human breast cancer cell line T47D and the human epidermal carcinoma cell A431 using RT-PCR and Southern blot analysis of the PCR product (Gorbulev et al., 1994). In the guinea-pig, expression was detected in the pregnant uterus and the brain (Gorbulev et al., 1992), whereas its expression in the rat was confined to the secondary spermatocytes when various tissues were examined by Northern blot analysis and in-situ hybridization (Fathi et al., 1993). This pattern is quite different from the other mammalian bombesin receptors, GRP-R and NMB-R, which are predominantly expressed in the CNS and GI tract ( Kroog et al., 1995). In contrast to the mouse GRP-R gene, whose transcription is regulated by cAMP and dexamethasone ( Weber et al., 1995), nothing is known about the mechanisms regulating BRS-3 gene expression. To gain insight in the structural basis of BRS-3 gene expression, we report in this paper the structure of the mouse BRS-3 gene and the chromosomal localization of the human and mouse BRS-3 gene.
2.3. Genomic clones Bacteriophage P1 genomic clones were obtained from Genome Systems, Inc. (St. Louis, MO) containing either the entire mouse (GS #5671) or human (DMPC-HFF #1-340-12B; GS #9463) BRS-3 gene. Two pairs of BRS-3 gene-specific synthetic oligonucleotides amplified BRS-3 specific products of either 330 bp size (mouse) or 351 bp (human) on an appropriate genomic DNA template. These two primer pairs, (1) mouse, sense: 5∞-GACAACTCTCCAGGAATAGA-3∞, anti-sense: 5∞-ACTGTCAGCGCTGAGAAT-3∞ and (2) human, sense: 5∞-GAGTATCTGGATGTCTTGGATTTTC3∞, anti-sense: 5∞-GCATGGATTTGGTCTTGAAAAAG-3∞, were then used to screen bacteriophage P1 genomic libraries (Genome Systems, Inc., St. Louis, MO). 2.4. DNA sequencing Large-scale P1 bacteriophage DNA was prepared according to the methods provided by the supplier (Genome Systems, Inc., St. Louis, MO) and 1 mg was used as template for each sequencing reaction. The 5∞ and 3∞ flanking regions, all three exons and exon/intron boundaries were sequenced using end-labeled, genespecific synthetic oligonucleotides and the fmol DNA sequencing kit (Promega, Madison, WI ) following the recommendations of the supplier. Subcloned P1 DNA fragments were sequenced using miniprep DNA templates ( Wizard@ miniprep DNA purification kit, Promega; Madison, WI ). 2.5. PCR
2. Material and methods 2.1. Chemicals Restriction and modifying enzymes were purchased from Gibco/BRL (Gaithersburg, MD) unless stated otherwise. Synthetic oligonucleotides were obtained from Midland ( The Midland Certified Reagent Company, Midland, TX ). Ten per cent (w/v) SDS (sodium dodecyl sulfate) was purchased from Quality Biological (Gaithersburg, MD). Sodium acetate solution (3 M, pH 5.2) was purchased from Advanced Biotechnologies Inc. (Columbia, MD).
2.2. Radioactive material [a-32P] dCTP (3000 Ci/mmol ) and [c-32P] ATP (3000 Ci/mmol ) were purchased from DuPont/NEN (Boston, MA).
Polymerase chain reactions (PCR) were carried out with the GeneAmp PCR System 9600 (Perkin Elmer Cetus, Emeryville, CA) using routine conditions and buffer provided by the manufacturer. Intronic sequences were amplified using the GeneAmp XL PCR kit (Perkin Elmer Cetus, Emeryville, CA). 2.6. Subcloning Intronic genomic DNA sequences from human and mouse BRS-3 genes were amplified by PCR using exonderived, gene-specific oligonucleotides ( The Midland Certified Reagent Company, Midland, TX ) and bacteriophage P1 genomic BRS-3 DNA as template. The size of the resulting PCR products was determined by agarose gel electrophoresis ( FMC BioProducts, Rockland, ME ), and they were either sequenced (fmol kit, Promega, Madison, WI ) directly or after subcloning into the plasmid vector pCR@II using the TA cloning kit (Invitrogen, San Diego, CA). Miniprep plasmid DNA ( Wizard@ miniprep DNA purification kit,
H.C. Weber et al. / Gene 211 (1998) 125–131
Promega, Madison, WI ) was digested with various restriction enzymes and separated electrophoretically in 0.5% (w/v) SeaKem GTG agarose gels (FMC BioProducts, Rockland, ME ). Recombinant clones were identified by restriction enzyme digestion, and miniprep DNA templates were then used to determine the nucleotide sequences across the exon/intron borders of the BRS-3 genes. 2.7. DNA sequence analysis Nucleotide and amino acid sequences were analyzed and aligned with the software package Gene WorksA (IntelliGenetics, Inc., Mountain View, CA). 2.8. Fluorescence in-situ hybridization (FISH) BRS-3 specific DNA from human (DMPC-HFF #1-340-12B; GS #9463) or mouse (GS #5671) bacteriophage P1 genomic clones was labeled with digoxigenin dUTP by nick translation, combined with sheared mouse or human DNA, respectively, and hybridized to either normal metaphase chromosomes derived from PHA stimulated blood lymphocytes (human) or from male embryonic stem cells (mouse) under conditions specified by the contractor (Genome Systems, Inc., St. Louis, MO). Specific hybridization signals were detected by incubating the slides with fluoresceinated antidigoxigenin antibodies and Texas red avidin followed by counterstaining with DAPI. A biotin labeled probe specific for the centromere of the human X-chromosome (DXZ1) or specific for the centromeric region of the mouse X-chromosome was hybridized together with the human (DMPC-HFF #1-340-12B; GS #9463) or mouse (GS #5671) clone, respectively. A total of 80 metaphase cells were analyzed with 73 (human) or 75 (mouse) exhibiting gene-specific labeling (Genome Systems, Inc., St. Louis, MO).
3. Results 3.1. Structure of the human and mouse BRS-3 genes Two bacteriophage P1 genomic clones harboring either the entire human or mouse BRS-3 gene were identified by PCR screening with gene-specific oligonucleotides. We determined the nucleotide sequence of the protein-coding regions, intron/exon borders and the 5∞ and 3∞ flanking regions of both genes using P1 genomic BRS-3 templates. The nucleotide sequence of the human BRS-3 gene (data not shown) was found to be identical to the data in a previously published report in which a clone derived from a human placental genomic library had been characterized (Gorbulev et al., 1994). The nucleotide sequence and structure of the mouse BRS-3
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gene is shown in Fig. 1. We also determined the intron sizes for both the human and the mouse BRS-3 genes by PCR and agarose gel electrophoresis (data not shown). Intron 1 in the human and mouse BRS-3 genes measured 1.5 kb and 1.6 kb, respectively (Fig. 1). The lengths of intron 2 were determined to be 1.3 kb in the human BRS-3 gene and 1.6 kb in the mouse homologue ( Fig. 1). The exon/intron splice sites occurred at residue 145 in the second intracellular loop and at residue 262 in the third intracellular loop, respectively ( Figs. 1 and 2). When the 5∞ flanking region of the mouse BRS-3 gene was searched for the presence of consensus sequences for cis-regulatory elements ( Faisst and Meyer, 1992), no binding motifs for SP-1, AP-1, and AP-2 were found. There are two TATA-like motifs present in the 5∞ flanking region of the mouse BRS-3 gene, located at 492–486 bp and more than 1 kb, respectively, upstream of the translation start codon ATG (Fig. 1). Comparing the nucleotide sequence of the 5∞ flanking regions of both the human and the mouse BRS-3 genes, we found an overall 58% homology within 1000 bp and 72% homology within the immediate 500 bp upstream of translation initiation codon ATG (data not shown). The open reading frame of the putative mouse BRS-3 gene is contained in three exons (Fig. 1) and can encode a protein of 399 amino acids (Fig. 2), which is identical in size compared to its human (399 residues) and guineapig (399 residues) homologues (Gorbulev et al., 1992, 1994; Fathi et al., 1993). The overall identity of the mouse BRS-3 protein sequence compared to human and guinea pig BRS-3 is 85% and 83%, respectively, with the carboxyl terminus being the most divergent protein sequence (Fig. 2). DNA sequencing of the carboxyl terminus of a cDNA clone confirmed the genomic DNA sequence (data not shown). A hydrophobicity plot shows a pattern compatible with seven transmembrane spanning domains in the mouse BRS-3 protein (data not shown). The putative mouse BRS-3 receptor has several potential consensus N-glycosylation (amino-acid numbers 10, 18, 29, 197, 303, and 364) and PKC-phosphorylation sites (amino-acid numbers 2, 32, 209, 265, and 391), respectively (Fig. 2).
3.2. Chromosomal localization Human and mouse BRS-3 specific P1 genomic DNA was used to determine their chromosomal localization using FISH ( Fig. 3). The human BRS-3 gene locus was mapped to chromosome band Xq25 ( Fig. 3), which confirms two previous reports assigning it to the X-chromosome ( Fathi et al., 1993; Gorbulev et al., 1994). Using the same technique (FISH ), we determined the mouse BRS-3 gene to map to the mouse X-chromosome XA7.1–7.2 ( Fig. 3).
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Fig. 1. Mouse BRS-3 gene nucleotide sequence. The protein coding region of the mouse BRS-3 gene is divided into three exons (boxed) and printed in upper-case letters. The nucleotide sequence of the 5∞ and 3∞ flanking regions and introns are printed in lower-case letters. The entire nucleotide sequence is numbered as indicated on the left side (deposited in GenBank under Accession Nos U 84899, U 84900, U 84901). The translation start codon ATG, the termination codon TAG, and two TATA-like motifs in the 5∞ flanking region are printed in bold. The intron sizes are indicated in kilobase pairs on the right-hand margin.
4. Discussion In this study, we report the cloning and structural organization of the mouse BRS-3 gene and the chromosomal localization of the mouse and human BRS-3 genes. The mouse BRS-3 gene demonstrates a significant
similarity to its human homologue and other mammalian bombesin receptor genes. Common to all mammalian bombesin receptor genes studied ( Weber et al., 1996), and in this study determined for the mouse BRS-3 gene ( Fig. 1), the exon/intron structure is conserved with the protein-coding region being contained
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Fig. 2. Amino acid alignment of Bombesin Receptor Subtype 3. The amino acid sequences of the mouse (399 residues), human (399 residues) and guinea-pig (399 residues) BRS-3 receptors are aligned using the computer software Gene WorksA (IntelliGenetics, Inc., Mountain View, CA). Identical residues for all three species are shaded. The seven putative transmembrane domains are indicated by overlining horizontal bars. Potential N-glycosylation sites (*) and PKC-phosphorylation sites ($) of the mouse BRS-3 are indicated above the amino acid sequence. The exon/intron splice sites are marked with a triangle (( ).
in three exons and the splice sites occurring in the proximal second and distal third intracellular loop with respect to the receptor protein. Intron sizes of the human and mouse BRS-3 genes are very similar; however, previously reported data for the human BRS-3 introns (intron 1, 1.1 kb; intron 2, 1.35 kb) (Gorbulev et al., 1994) differ slightly from ours, which is likely attributable to differences in the method of measurement. When the nucleotide sequences of the immediate 5∞ flanking regions of the human and mouse BRS-3 genes are aligned, a significantly higher degree of homology is found than when compared to other genes of the mammalian bombesin receptor gene family ( Weber et al., 1996). As seen in our study of the human BRS-3 gene and in a previous report (Gorbulev et al., 1994), no cisregulatory recognition sites for SP-1, AP-1, and AP-2 were found, but two TATA-like motifs are observed in the mouse BRS-3 promoter region. Since the transcription start sites have not yet been mapped for either the human or the mouse BRS-3 gene, the role for either TATA-motif in transcription remains unclear. A comparison of the putative mouse BRS-3 protein with other members of the mammalian bombesin receptor subfami-
lies demonstrates a 48% amino acid identity with the mouse GRP-R and 46% with the mouse NMB-R (data not shown) with the highest degree of homology residing in the transmembrane domains III and VII. At present, however, the exact physiological function of BRS-3 remains obscure. Localization of the human BRS-3 locus by FISH to chromosomal band Xq25 maps it to a slightly different position than band Xq26–28, reported previously (Gorbulev et al., 1994), where localization was determined by PCR analysis of DNA from human–mouse somatic cell hybrids. Our data position the human BRS-3 gene closer to the locus of the Lowe oculocerebrorenal syndrome (OCRL) whose gene was initially placed by genetic and physical mapping to Xq24–26 ( Wadelius et al., 1989; Reilly et al., 1990) and, more recently, to a X-chromosome breakpoint occurring in one patient localized at Xq26.1 (Mueller et al., 1991). An open reading frame encoding the putative OCRL gene (Attree et al., 1992) is clearly distinct from human BRS-3. The human GRP-R locus has been mapped to the short arm of the X-chromosome ( Xp22) (Maslen and Boyd, 1993; Weber et al., 1996) and, therefore, its
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among mammalian X-chromosomes, suggesting that their respective rat homologues are probably also localized on the X-chromosome.
References
Fig. 3. Chromosomal localization of the human and mouse BRS-3 genes. BRS-3 gene-specific labeled P1 genomic DNA was used to perform fluorescence in-situ hybridization (FISH ) on normal human (A, left) or mouse (B, right), respectively, metaphase chromosomes as described in Materials and methods. Gene-specific labeling of 73 human metaphase cells mapped the human BRS-3 gene locus (indicated by arrow) to chromosome band Xq25 (left panel ), and genespecific labeling of 75 mouse metaphase cells mapped the mouse BRS-3 gene locus (indicated by arrow) to chromosome band XA7.1–7.2 (right panel ).
locus is not linked to the human BRS-3 gene. We determined the mouse BRS-3 gene to map to the chromosomal band XA7.1–7.2 (Fig. 3), which is localized in the vicinity of the genes for the hypoxanthine guanine phosphoribosyl transferase (HPRT ) (Derry and Barnard, 1991; Herman et al., 1996), coagulation factor IX (Herman et al., 1996), and cerebellar degeneration related protein (Herman et al., 1996) but clearly distinct from the mouse GRP-R gene mapped to band XF3 (Maslen and Boyd, 1993; Weber et al., 1996). The GRP-R and BRS-3 genes in human and mouse represent, therefore, another example of synteny of genes
Alexander, R.W., Upp, J.R., Jr, Poston, G.J., Gupta, V., Townsend, C.M., Jr, Thompson, J.C., 1988. Effects of bombesin on growth of human small cell lung carcinoma in vivo. Cancer Res. 48, 1439–1441. Attree, O., Olivos, I.M., Okabe, I., Bailey, L.C., Nelson, D.L., Lewis, R.A., McInnes, R.R., Nussbaum, R.L., 1992. The Lowe’s oculocerebrorenal syndrome gene encodes a protein highly homologous to inositol polyphosphate-5-phosphatase. Nature 358, 239–242. Battey, J.F., Way, J.M., Corjay, M.H., Shapira, H., Kusano, K., Harkins, R., Wu, J.M., Slattery, T., Mann, E., Feldman, R.I., 1991. Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA 88, 395–399. Bold, R.J., Lowry, P.S., Ishizuka, J., Battey, J.F., Townsend, C.M., Jr, Thompson, J.C., 1994. Bombesin stimulates the in vitro growth of a human gastric cancer cell line. J. Cell. Physiol. 161, 519–525. Bologna, M., Festuccia, C., Muzi, P., Biordi, L., Ciomei, M., 1989. Bombesin stimulates growth of human prostatic cancer cells in vitro. Cancer 63, 1714–1720. Carney, D.N., Cuttitta, F., Moody, T.W., Minna, J.D., 1987. Selective stimulation of small cell lung cancer clonal growth by bombesin and gastrin-releasing peptide. Cancer Res. 47, 821–825. Corjay, M.H., Dobrzanski, D.J., Way, J.M., Viallet, J., Shapira, H., Worland, P., Sausville, E.A., Battey, J.F., 1991. Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells. J. Biol. Chem. 266, 18771–18779. Cuttitta, F., Carney, D.N., Mulshine, J., Moody, T.W., Fedorko, J., Fischler, A., Minna, J.D., 1985. Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer cells. Nature 316, 823–826. Derry, J.M., Barnard, P.J., 1991. Mapping of the glycine receptor a2-subunit gene and the GABA a3-subunit gene on the mouse X A chromosome. Genomics 10, 593–597. Erspamer, V., Erspamer, G.F., Inselvini, M., Negri, L., 1972. Occurrence of bombesin and alytesin in extracts of the skin of three European discoglossid frogs and pharmacological actions of bombesin on extravascular smooth muscle. Br. J. Pharmacol. 45, 333–348. Erspamer, V., 1988. Discovery, isolation and characterization of bombesin-like peptides. Ann. NY Acad. Sci. 547, 3–9. Faisst, S., Meyer, S., 1992. Compilation of vertebrate-encoded transcription factors. Nucleic Acids Res. 20, 3–26. Fathi, Z., Corjay, M.H., Shapira, H., Wada, E., Benya, R., Jensen, R., Viallet, J., Sausville, E.A., Battey, J.F., 1993. BRS-3: novel bombesin receptor subtype selectively expressed in testis and lung carcinoma cells. J. Biol. Chem. 268 (#8), 5979–5984. Ghatei, M.A., Jung, R.T., Stevenson, J.C., Hillyard, C.J., Adrian, T.E., Lee, Y.C., Christofides, N.D., Sarson, D.L., Mashiter, K., MacIntyre, I., Bloom, S.R., 1982. Bombesin action on gut hormones and calcium in man. J. Clin. Endocrinol. Metab. 54, 980–985. Gorbulev, V., Akhundova, A., Buchner, H., Fahrenholz, F., 1992. Molecular cloning of a new bombesin receptor subtype expressed in uterus during pregnancy. Eur. J. Biochem. 208, 405–410. Gorbulev, V., Akhundova, A., Grzeschik, K.H., Fahrenholz, F., 1994. Organization and chromosomal localization of the gene for the human bombesin receptor subtype expressed in pregnant uterus. FEBS Lett. 340, 260–264. Herman, G.E., Blair, H.J., Boyd, Y., Brown, S.D.M., de Gouyon, B., Haynes, A., Quaderi, N., 1996. Mouse X chromosome. Mammal. Genome 6, S317–S330.
H.C. Weber et al. / Gene 211 (1998) 125–131 Kroog, G.S., Jensen, R.T., Battey, J.F., 1995. Mammalian bombesin receptors. Med. Res. Rev. 15, 389–417. Lebacq-Verheyden, A.M., Trepel, J., Sausville, E.A., Battey, J.F., 1990. Peptide growth factors and their receptors. Handb. Exp. Pharmacol. 95, 71–124. Maslen, G.L., Boyd, Y., 1993. Comparative mapping of the Grpr locus on the X chromosomes of man and mouse. Genomics 17, 106–109. Mueller, O.T., Hartsfield, J.K., Jr, Gallardo, L.A., Essig, Y.P., Miller, K.L., Papenhausen, P.R., Tedesco, T.A., 1991. Lowe oculocerebrorenal syndrome in a female with a balanced X;20 translocation: mapping of the X chromosome breakpoint. Am. J. Hum. Genet. 49, 804–810. Narayan, S., Spindel, E.R., Rubin, N.H., Singh, P., 1992. A potent bombesin receptor antagonist inhibits bombesin-stimulated growth of mouse colon cancer cells in vitro: absence of autocrine effects. Cell Growth Differ. 3, 111–118. Nelson, J., Donnelly, M., Walker, B., Gray, J., Shaw, C., Murphy, R.F., 1991. Bombesin stimulates proliferation of human breast cancer cells in culture. Br. J. Cancer 63, 933–936. Reilly, D.S., Lewis, R.A., Nussbaum, R.L., 1990. Genetic and physical mapping of Xq24–q26 markers flanking the Lowe oculocerebrorenal syndrome. Genomics 8, 62–70. Severi, C., Jensen, R.T., Erspamer, V., D’Arpino, L., Coy, D.H.,
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Torsoli, A., Delle Fave, G., 1991. Different receptors mediate the action of bombesin-related peptides on gastric smooth muscle cells. Am. J. Physiol. 260, G683–G690. Spindel, E.R., Giladi, E., Brehm, P., Goodman, R.H., Segerson, T.P., 1990. Cloning and functional characterization of a complementary DNA encoding the murine fibroblast bombesin/gastrin-releasing peptide receptor. Mol. Endocrinol. 4, 1956–1963. Wadelius, C., Fagerholm, P., Pettersoon, U., Anneren, G., 1989. Lowe oculocerebrorenal syndrome: DNA-based linkage of the gene to Xq24–q26, using tightly linked flanking markers and the correlation to lens examination in carrier diagnosis. Am. J. Hum. Genet. 44, 241–247. Weber, H.C., Mrozinski, J.E., Jensen, R.T., Battey, J.F., 1995. Molecular structure and regulation of the expression by dexamethasone and cAMP of the gastrin-releasing peptide receptor gene. Gastroenterology (Suppl.) 108, A1015 Weber, H.C., Hampton, L.L., Jensen, R.T., Battey, J.F., 1996. Structure and chromosomal localization of the genes for the gastrinreleasing peptide receptor, neuromedin-B receptor and the bombesin subtype 3 receptor. Regul. Pept. 64, 207 Weber, S., Zuckerman, J.E., Bostwich, D.G., Bensch, K.G., Sikic, B.I., Raffin, T.A., 1985. Gastrin releasing peptide is a selective mitogen for small cell lung carcinoma in vitro. J. Clin. Invest. 75, 306–309.
Structure and chromosomal localization of the mouse bombesin receptor subtype 3 gene H.C. Weber a,*, L.L. Hampton b, R.T. Jensen c, J.F. Battey b a Section of Gastroenterology, Boston University School of Medicine, 88 East Newton Street, Boston, MA 02118-2393, USA b National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, MD 20850, USA c Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA Received 3 July 1997; received in revised form 30 January 1998; accepted 2 February 1998; Received by A. Dugaiczyk
Abstract Bombesin (BN )-like peptides/neurotransmitters mediate a broad range of physiological funtions in the gastrointestinal tract and the central nervous system through binding to their specific, high-affinity mammalian bombesin receptors. This family of heptahelical, G protein-coupled receptors includes the gastrin-releasing peptide receptor (GRP-R, or bb2), neuromedin B receptor (NMB-R, or bb1), and the bombesin receptor subtype 3 (BRS-3, or bb3). The tissue distribution of BRS-3 is quite dissimilar compared to the other two BN receptors, GRP-R and NMB-R, and a natural ligand for BRS-3 is currently unknown. Nothing is known about mechanisms regulating BRS-3 gene expression and possible association with disease. To gain insight into the underlying structure and chromosomal localization of the BRS-3 genes, bacteriophage P1 genomic clones, harboring the genes for the human and mouse BRS-3, respectively, were isolated and their structure and chromosomal localizations determined. The protein-coding region of both genes is divided into three exons and spans approximately 5 kb. The loci of the BRS-3 genes were mapped to a syntenic region of the human ( Xq25) and mouse ( XA7.1–7.2) X-chromosome, respectively. The structural data of the BRS-3 genes derived from this study will permit future investigations of the mechanisms regulating their expression. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Gene structure; Mouse X-chromosome; Human X-chromosome; Genomic clones; Mammalian; G protein-coupled receptors
1. Introduction The bombesin (BN )-like peptides/neurotransmitters mediate a variety of biological activities in the central nervous system (CNS) and gastrointestinal (GI ) tract, including smooth muscle contraction and secretion of other GI hormones (Lebacq-Verheyden et al., 1990; Severi et al., 1991; Ghatei et al., 1982). They have also been implicated as autocrine growth factors in the * Corresponding author. Tel: +1 617 638 6547; Fax: +1 617 638 7785; e-mail: [email protected] Abbreviations: BN, bombesin; bp, base pair(s); BRS-3, bombesin receptor subtype 3; DAPI, 4∞, 6-diamidino-2-phenylindole; FISH, fluorescence in-situ hybridization; GRP, gastrin-releasing peptide; GRP-R, gastrin-releasing peptide receptor; kb, kilobase pairs; NMB, neuromedin B; NMB-R, neuromedin B receptor; PCR, polymerase chain reaction; PHA, phytohaemagglutinin; SCLC, small cell lung carcinoma. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 05 0 - X
pathogenesis and progression of some human small cell lung carcinomas (Cuttitta et al., 1985) and the stimulation of proliferation of breast, colon, gastric, small cell lung cancer and prostate cancer cells in vitro or in vivo ( Weber et al., 1985; Carney et al., 1987; Alexander et al., 1988; Bologna et al., 1989; Nelson et al., 1991; Narayan et al., 1992; Bold et al., 1994). The amidated tetradecapeptide bombesin (BN ) has been isolated originally from the skin of the European frog Bombina bombina ( Erspamer et al., 1972), and to date, two mammalian BN-like peptides, the gastrin-releasing peptide (GRP) and neuromedin B (NMB), have been isolated and characterized ( Erspamer, 1988). BN-like peptides mediate their wide spectrum of functions by binding to specific, high-affinity cell surface receptors that belong to the superfamily of heptahelical G protein-coupled receptors. The receptors for the mammalian peptides GRP and NMB have been characterized, and their cDNAs have been isolated from human and murine
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tissues (Spindel et al., 1990; Battey et al., 1991; Corjay et al., 1991). Much less is known about the third member of this receptor family, the bombesin receptor subtype-3 (BRS-3) whose natural ligand is unknown. Human BRS-3 cDNA was isolated from a small cell lung carcinoma (SCLC ) cell line, and is expressed in a panel of human lung carcinoma cell lines (Fathi et al., 1993), and in the pregnant human myometrium (Gorbulev et al., 1994). In addition, BRS-3 mRNA was also expressed at a very low abundance in the human breast cancer cell line T47D and the human epidermal carcinoma cell A431 using RT-PCR and Southern blot analysis of the PCR product (Gorbulev et al., 1994). In the guinea-pig, expression was detected in the pregnant uterus and the brain (Gorbulev et al., 1992), whereas its expression in the rat was confined to the secondary spermatocytes when various tissues were examined by Northern blot analysis and in-situ hybridization (Fathi et al., 1993). This pattern is quite different from the other mammalian bombesin receptors, GRP-R and NMB-R, which are predominantly expressed in the CNS and GI tract ( Kroog et al., 1995). In contrast to the mouse GRP-R gene, whose transcription is regulated by cAMP and dexamethasone ( Weber et al., 1995), nothing is known about the mechanisms regulating BRS-3 gene expression. To gain insight in the structural basis of BRS-3 gene expression, we report in this paper the structure of the mouse BRS-3 gene and the chromosomal localization of the human and mouse BRS-3 gene.
2.3. Genomic clones Bacteriophage P1 genomic clones were obtained from Genome Systems, Inc. (St. Louis, MO) containing either the entire mouse (GS #5671) or human (DMPC-HFF #1-340-12B; GS #9463) BRS-3 gene. Two pairs of BRS-3 gene-specific synthetic oligonucleotides amplified BRS-3 specific products of either 330 bp size (mouse) or 351 bp (human) on an appropriate genomic DNA template. These two primer pairs, (1) mouse, sense: 5∞-GACAACTCTCCAGGAATAGA-3∞, anti-sense: 5∞-ACTGTCAGCGCTGAGAAT-3∞ and (2) human, sense: 5∞-GAGTATCTGGATGTCTTGGATTTTC3∞, anti-sense: 5∞-GCATGGATTTGGTCTTGAAAAAG-3∞, were then used to screen bacteriophage P1 genomic libraries (Genome Systems, Inc., St. Louis, MO). 2.4. DNA sequencing Large-scale P1 bacteriophage DNA was prepared according to the methods provided by the supplier (Genome Systems, Inc., St. Louis, MO) and 1 mg was used as template for each sequencing reaction. The 5∞ and 3∞ flanking regions, all three exons and exon/intron boundaries were sequenced using end-labeled, genespecific synthetic oligonucleotides and the fmol DNA sequencing kit (Promega, Madison, WI ) following the recommendations of the supplier. Subcloned P1 DNA fragments were sequenced using miniprep DNA templates ( Wizard@ miniprep DNA purification kit, Promega; Madison, WI ). 2.5. PCR
2. Material and methods 2.1. Chemicals Restriction and modifying enzymes were purchased from Gibco/BRL (Gaithersburg, MD) unless stated otherwise. Synthetic oligonucleotides were obtained from Midland ( The Midland Certified Reagent Company, Midland, TX ). Ten per cent (w/v) SDS (sodium dodecyl sulfate) was purchased from Quality Biological (Gaithersburg, MD). Sodium acetate solution (3 M, pH 5.2) was purchased from Advanced Biotechnologies Inc. (Columbia, MD).
2.2. Radioactive material [a-32P] dCTP (3000 Ci/mmol ) and [c-32P] ATP (3000 Ci/mmol ) were purchased from DuPont/NEN (Boston, MA).
Polymerase chain reactions (PCR) were carried out with the GeneAmp PCR System 9600 (Perkin Elmer Cetus, Emeryville, CA) using routine conditions and buffer provided by the manufacturer. Intronic sequences were amplified using the GeneAmp XL PCR kit (Perkin Elmer Cetus, Emeryville, CA). 2.6. Subcloning Intronic genomic DNA sequences from human and mouse BRS-3 genes were amplified by PCR using exonderived, gene-specific oligonucleotides ( The Midland Certified Reagent Company, Midland, TX ) and bacteriophage P1 genomic BRS-3 DNA as template. The size of the resulting PCR products was determined by agarose gel electrophoresis ( FMC BioProducts, Rockland, ME ), and they were either sequenced (fmol kit, Promega, Madison, WI ) directly or after subcloning into the plasmid vector pCR@II using the TA cloning kit (Invitrogen, San Diego, CA). Miniprep plasmid DNA ( Wizard@ miniprep DNA purification kit,
H.C. Weber et al. / Gene 211 (1998) 125–131
Promega, Madison, WI ) was digested with various restriction enzymes and separated electrophoretically in 0.5% (w/v) SeaKem GTG agarose gels (FMC BioProducts, Rockland, ME ). Recombinant clones were identified by restriction enzyme digestion, and miniprep DNA templates were then used to determine the nucleotide sequences across the exon/intron borders of the BRS-3 genes. 2.7. DNA sequence analysis Nucleotide and amino acid sequences were analyzed and aligned with the software package Gene WorksA (IntelliGenetics, Inc., Mountain View, CA). 2.8. Fluorescence in-situ hybridization (FISH) BRS-3 specific DNA from human (DMPC-HFF #1-340-12B; GS #9463) or mouse (GS #5671) bacteriophage P1 genomic clones was labeled with digoxigenin dUTP by nick translation, combined with sheared mouse or human DNA, respectively, and hybridized to either normal metaphase chromosomes derived from PHA stimulated blood lymphocytes (human) or from male embryonic stem cells (mouse) under conditions specified by the contractor (Genome Systems, Inc., St. Louis, MO). Specific hybridization signals were detected by incubating the slides with fluoresceinated antidigoxigenin antibodies and Texas red avidin followed by counterstaining with DAPI. A biotin labeled probe specific for the centromere of the human X-chromosome (DXZ1) or specific for the centromeric region of the mouse X-chromosome was hybridized together with the human (DMPC-HFF #1-340-12B; GS #9463) or mouse (GS #5671) clone, respectively. A total of 80 metaphase cells were analyzed with 73 (human) or 75 (mouse) exhibiting gene-specific labeling (Genome Systems, Inc., St. Louis, MO).
3. Results 3.1. Structure of the human and mouse BRS-3 genes Two bacteriophage P1 genomic clones harboring either the entire human or mouse BRS-3 gene were identified by PCR screening with gene-specific oligonucleotides. We determined the nucleotide sequence of the protein-coding regions, intron/exon borders and the 5∞ and 3∞ flanking regions of both genes using P1 genomic BRS-3 templates. The nucleotide sequence of the human BRS-3 gene (data not shown) was found to be identical to the data in a previously published report in which a clone derived from a human placental genomic library had been characterized (Gorbulev et al., 1994). The nucleotide sequence and structure of the mouse BRS-3
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gene is shown in Fig. 1. We also determined the intron sizes for both the human and the mouse BRS-3 genes by PCR and agarose gel electrophoresis (data not shown). Intron 1 in the human and mouse BRS-3 genes measured 1.5 kb and 1.6 kb, respectively (Fig. 1). The lengths of intron 2 were determined to be 1.3 kb in the human BRS-3 gene and 1.6 kb in the mouse homologue ( Fig. 1). The exon/intron splice sites occurred at residue 145 in the second intracellular loop and at residue 262 in the third intracellular loop, respectively ( Figs. 1 and 2). When the 5∞ flanking region of the mouse BRS-3 gene was searched for the presence of consensus sequences for cis-regulatory elements ( Faisst and Meyer, 1992), no binding motifs for SP-1, AP-1, and AP-2 were found. There are two TATA-like motifs present in the 5∞ flanking region of the mouse BRS-3 gene, located at 492–486 bp and more than 1 kb, respectively, upstream of the translation start codon ATG (Fig. 1). Comparing the nucleotide sequence of the 5∞ flanking regions of both the human and the mouse BRS-3 genes, we found an overall 58% homology within 1000 bp and 72% homology within the immediate 500 bp upstream of translation initiation codon ATG (data not shown). The open reading frame of the putative mouse BRS-3 gene is contained in three exons (Fig. 1) and can encode a protein of 399 amino acids (Fig. 2), which is identical in size compared to its human (399 residues) and guineapig (399 residues) homologues (Gorbulev et al., 1992, 1994; Fathi et al., 1993). The overall identity of the mouse BRS-3 protein sequence compared to human and guinea pig BRS-3 is 85% and 83%, respectively, with the carboxyl terminus being the most divergent protein sequence (Fig. 2). DNA sequencing of the carboxyl terminus of a cDNA clone confirmed the genomic DNA sequence (data not shown). A hydrophobicity plot shows a pattern compatible with seven transmembrane spanning domains in the mouse BRS-3 protein (data not shown). The putative mouse BRS-3 receptor has several potential consensus N-glycosylation (amino-acid numbers 10, 18, 29, 197, 303, and 364) and PKC-phosphorylation sites (amino-acid numbers 2, 32, 209, 265, and 391), respectively (Fig. 2).
3.2. Chromosomal localization Human and mouse BRS-3 specific P1 genomic DNA was used to determine their chromosomal localization using FISH ( Fig. 3). The human BRS-3 gene locus was mapped to chromosome band Xq25 ( Fig. 3), which confirms two previous reports assigning it to the X-chromosome ( Fathi et al., 1993; Gorbulev et al., 1994). Using the same technique (FISH ), we determined the mouse BRS-3 gene to map to the mouse X-chromosome XA7.1–7.2 ( Fig. 3).
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Fig. 1. Mouse BRS-3 gene nucleotide sequence. The protein coding region of the mouse BRS-3 gene is divided into three exons (boxed) and printed in upper-case letters. The nucleotide sequence of the 5∞ and 3∞ flanking regions and introns are printed in lower-case letters. The entire nucleotide sequence is numbered as indicated on the left side (deposited in GenBank under Accession Nos U 84899, U 84900, U 84901). The translation start codon ATG, the termination codon TAG, and two TATA-like motifs in the 5∞ flanking region are printed in bold. The intron sizes are indicated in kilobase pairs on the right-hand margin.
4. Discussion In this study, we report the cloning and structural organization of the mouse BRS-3 gene and the chromosomal localization of the mouse and human BRS-3 genes. The mouse BRS-3 gene demonstrates a significant
similarity to its human homologue and other mammalian bombesin receptor genes. Common to all mammalian bombesin receptor genes studied ( Weber et al., 1996), and in this study determined for the mouse BRS-3 gene ( Fig. 1), the exon/intron structure is conserved with the protein-coding region being contained
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Fig. 2. Amino acid alignment of Bombesin Receptor Subtype 3. The amino acid sequences of the mouse (399 residues), human (399 residues) and guinea-pig (399 residues) BRS-3 receptors are aligned using the computer software Gene WorksA (IntelliGenetics, Inc., Mountain View, CA). Identical residues for all three species are shaded. The seven putative transmembrane domains are indicated by overlining horizontal bars. Potential N-glycosylation sites (*) and PKC-phosphorylation sites ($) of the mouse BRS-3 are indicated above the amino acid sequence. The exon/intron splice sites are marked with a triangle (( ).
in three exons and the splice sites occurring in the proximal second and distal third intracellular loop with respect to the receptor protein. Intron sizes of the human and mouse BRS-3 genes are very similar; however, previously reported data for the human BRS-3 introns (intron 1, 1.1 kb; intron 2, 1.35 kb) (Gorbulev et al., 1994) differ slightly from ours, which is likely attributable to differences in the method of measurement. When the nucleotide sequences of the immediate 5∞ flanking regions of the human and mouse BRS-3 genes are aligned, a significantly higher degree of homology is found than when compared to other genes of the mammalian bombesin receptor gene family ( Weber et al., 1996). As seen in our study of the human BRS-3 gene and in a previous report (Gorbulev et al., 1994), no cisregulatory recognition sites for SP-1, AP-1, and AP-2 were found, but two TATA-like motifs are observed in the mouse BRS-3 promoter region. Since the transcription start sites have not yet been mapped for either the human or the mouse BRS-3 gene, the role for either TATA-motif in transcription remains unclear. A comparison of the putative mouse BRS-3 protein with other members of the mammalian bombesin receptor subfami-
lies demonstrates a 48% amino acid identity with the mouse GRP-R and 46% with the mouse NMB-R (data not shown) with the highest degree of homology residing in the transmembrane domains III and VII. At present, however, the exact physiological function of BRS-3 remains obscure. Localization of the human BRS-3 locus by FISH to chromosomal band Xq25 maps it to a slightly different position than band Xq26–28, reported previously (Gorbulev et al., 1994), where localization was determined by PCR analysis of DNA from human–mouse somatic cell hybrids. Our data position the human BRS-3 gene closer to the locus of the Lowe oculocerebrorenal syndrome (OCRL) whose gene was initially placed by genetic and physical mapping to Xq24–26 ( Wadelius et al., 1989; Reilly et al., 1990) and, more recently, to a X-chromosome breakpoint occurring in one patient localized at Xq26.1 (Mueller et al., 1991). An open reading frame encoding the putative OCRL gene (Attree et al., 1992) is clearly distinct from human BRS-3. The human GRP-R locus has been mapped to the short arm of the X-chromosome ( Xp22) (Maslen and Boyd, 1993; Weber et al., 1996) and, therefore, its
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among mammalian X-chromosomes, suggesting that their respective rat homologues are probably also localized on the X-chromosome.
References
Fig. 3. Chromosomal localization of the human and mouse BRS-3 genes. BRS-3 gene-specific labeled P1 genomic DNA was used to perform fluorescence in-situ hybridization (FISH ) on normal human (A, left) or mouse (B, right), respectively, metaphase chromosomes as described in Materials and methods. Gene-specific labeling of 73 human metaphase cells mapped the human BRS-3 gene locus (indicated by arrow) to chromosome band Xq25 (left panel ), and genespecific labeling of 75 mouse metaphase cells mapped the mouse BRS-3 gene locus (indicated by arrow) to chromosome band XA7.1–7.2 (right panel ).
locus is not linked to the human BRS-3 gene. We determined the mouse BRS-3 gene to map to the chromosomal band XA7.1–7.2 (Fig. 3), which is localized in the vicinity of the genes for the hypoxanthine guanine phosphoribosyl transferase (HPRT ) (Derry and Barnard, 1991; Herman et al., 1996), coagulation factor IX (Herman et al., 1996), and cerebellar degeneration related protein (Herman et al., 1996) but clearly distinct from the mouse GRP-R gene mapped to band XF3 (Maslen and Boyd, 1993; Weber et al., 1996). The GRP-R and BRS-3 genes in human and mouse represent, therefore, another example of synteny of genes
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